ES2233955T3 - Adjuvants for viral vaccines. - Google Patents

Abstract

THIS INVENTION DESCRIBES COMPOSITIONS OF VACCINES FOR MAMMALS CONTAINING AN EFFECTIVE AMOUNT OF AN ASSISTANT (SCUALENO OR SCALAN), GLICEROL AND A SURFACTANT. OPTIONALLY, THESE COMPOSITIONS MAY CONTAIN ALUMINUM SALTS AND ONE OR MORE PHARMACEUTICALLY ACCEPTABLE STAMPS.

Description

Adjuvants for viral vaccines.

The present invention relates to new adjuvants that are useful for increasing the antigenicity of mammalian vaccines, and mammalian vaccines containing adjuvants More particularly, the present invention relates to new adjuvants for vaccines using squalene or squalene, or mixtures thereof, glycerol and a surfactant.

Four main categories of vaccines to generate antibodies against mammalian diseases, particularly in humans. These are vaccines with live microbes, vaccines with dead or inactivated microbes, vaccines with subunits and toxoids. Of this group, vaccines with Live microbes produce the most potent immune response in the host. These live microbes vaccines are usually attenuated, so that they are able to establish a prolonged immune response to its antigens without causing disease with which are normally associated.

Vaccines with dead microbes are inactivated with chemicals or other means that do not inactivate the factors antigens that present to the host's immune system. The whooping cough vaccine and the Salk vaccine against Polio are examples of vaccines with dead microbes.

In the case of some disease vectors, even killing the organism does not prevent them from causing effects undesirable in the recipient. In such cases, the agent must fragment into subunits or subfractions that are not, per se same, pathogenic. Examples of such vaccines with subunits include some flu shots and some shots experimental against the herpes virus and against hepatitis.

Finally, toxoid vaccines are those in which a toxin excreted by an organism is transformed into no toxic and then used like any other antigenic factor to generate an immune response against the toxin. The toxoid Tetanus is an example of such a vaccine.

Unfortunately, vaccines with subunits and toxoids rarely generate enough antigens to trigger prolonged immunity in the host. Therefore, immunity induced with vaccines with non-live microbes, and especially that of Vaccines with highly purified subunits, lasts only one to two years.

Several adjuvants have been described to reinforce the immunogenicity of vaccines with non-live microbes. Between they are the complete Freund's adjuvant, which includes oil mineral, water and emulsifier and dead tuberculosis bacteria. Of the many adjuvants available for use in mammals, only a few, including aluminum hydroxide and aluminum phosphate, have been widely used in beings humans. Various adjuvants have been rejected for use in humans because they cause local or systemic reactions serious. Some of them, like Freund's complete adjuvant that It contains mineral oil, they are not metabolizable and, because cause cancer in laboratory animals, are considered possible carcinogens Therefore, effective adjuvants and insurance capable of enhancing the action of vaccines, especially of vaccines of the type of subunits that are currently available and those that can be developed.

Therefore, according to the present invention provides a mammalian vaccine composition comprising a inactivated vaccine complete or in subunits and an effective amount of an adjuvant, characterized in that the adjuvant comprises squalene or squalane, or a mixture thereof, glycerol and a surfactant

According to an additional aspect of the present invention, an adjuvant comprising squalene or Squalane, or a mixture thereof, glycerol and a surfactant

The adjuvant of the present invention comprises squalene and / or squalene, glycerol and a surfactant. Plus particularly, the squalene and / or squalane component comprises from 1.2% to 47.6% of the adjuvant mixture, the component of glycerol comprises from 5.9% to 47.6% of the adjuvant mixture, and the surfactant component comprises from 0.12% to 4.8% of the adjuvant mixture. The percentages mentioned herein memory refer to the percentage by volume of each component, and it will be understood that the remaining percentage of the adjuvant mixture is generated by the other components described herein memory.

More particularly, when the adjuvant is in mixture form with a mammalian vaccine, the component of squalene and / or squalene comprises from about 1% and the approximately 40% of the total mixture, the glycerol component it comprises from about 5% and about 40% of the total mixture, and the surfactant component comprises from approximately 0.1% and approximately 4.0% of the total mixture. The percentages mentioned herein refer to the volume percentage of each component, and it will be understood that the Remaining percentage of the total mixture is generated by the vaccine, by itself, or by the vaccine and aluminum salts and buffers Optional described herein.

In a more preferred aspect of the present invention, the squalene and / or squalane component of the vaccine mammal comprises from 2% and 20% of the total mixture, glycerol it comprises from 10% and 30% of the total mixture, and the surfactant It comprises from 0.15% and 1.6% of the total mixture. In one aspect even more preferred of the present invention the component of squalene and / or squalene of the mammalian vaccine comprises from 3% and 7% of the total mixture, the glycerol component comprises from 15% and 25% of the total mixture, and the component of surfactant comprises from 0.18% and 0.22% of the mixture total.

With the term squalene the compound is indicated described on page 1383 of the Merck Index, 11th Edition, as (all-E) -2,6,10,15,19,23-hexamethyl-2,6, -10,14,18,22-tetracosahexane, as well as by the names sinacene and supraeno. Squalane, too mentioned on page 1383 of the Merck Index, 11th Edition, as 2,6,10,15,19,23-hexamethyltetracosan; perhydrosqualene; dodecahydrosqualene and spinacan, can be used instead of, or in mixtures with, the squalene component of the mixtures of adjuvants described herein.

Surfactants that can be used with present formulations include, but are not limited to, polysorbate 20 (polyoxyethylene 20 sorbitan monolaurate), Polysorbate 60, Span 80® Sorbitan Oleate, a product of ICI Americas, Wilmington, DE, Cremophor® surfactants manufactured by BASF Corporation, Parsippany, NJ, and polysorbate 80, which known as derivatives mono-9-octadecenoate poly (oxy-1,2-ethanediyl) of sorbitan, polyoxyethylene monooleate (20) sorbitan, polyoxyethylene of sorbitan monooleate, Sorlate, Tween 80, among others, and is indicative of a sorbitol oleate ester and its anhydrides copolymerized with approximately 20 moles of ethylene oxide per each mole of sorbitol and sorbitol anhydrides. Polysorbate 80 it is the preferred surfactant for use herein invention.

Those skilled in the art will appreciate that the glycerol component of the adjuvants herein are refers to the alcohol available in the market and also known as 1,2,3-propanotriol, glycerol, glycerin or trihydroxypropane.

Optionally, the adjuvants herein invention may also contain one or more aluminum salts, pharmaceutically acceptable, such as aluminum hydroxide, aluminum phosphate or aluminum sulfate, a a concentration in weight / volume from 0.1% and 2.0%, preferably from 0.3% and 0.7%, most preferably 0.5%. Aluminum hydroxide is the preferred aluminum salt.

The adjuvants of the present invention also may optionally contain one or more acceptable pH buffers from a pharmaceutical point of view, such as saline solution phosphate buffered (PBS), Tris-HCl, buffer citrate phosphate, Tricine buffer, Hepes buffers and maleate Other salts, such as KCl, can replace the chloride of sodium in the PBS buffer if the final solution remains substantially isotonic These buffers are preferably used to maintain the adjuvant mixture at a pH between 6.0 and 8.0.

Therefore, according to a more preferred aspect of the present invention a vaccine composition of mammals comprising an inactivated vaccine, whole or in subunits, and an effective amount of an adjuvant in a buffer pharmaceutically acceptable, characterized in that the adjuvant comprises squalene in the amount between the 3% and 7% by volume of the total composition, glycerol in the amount between 15% and 25% by volume of the total composition, a surfactant in the amount between 0.18% and 0.22% of the total composition, and between 0.1% and 2.0% of an aluminum salt.

The adjuvants of the present invention are preferably prepared with sterile components or sterilized using known thermal or filtration media. The mixture of adjuvant can be stored at 4 ° C. If the adjuvant is not sterile, no conservation at 4 ° C is recommended for more than 3 days. The adjuvant stock solutions of the present invention in Microfluidized, sonicated or homogenized form can be preserved frozen at a temperature, preferably of -20 ° C or lower.

The adjuvants used in the composition of the The present invention has a remarkable immunomodulatory activity, and it is believed that the adjuvant effect of these compounds is a direct result of its ability to modulate the response immune Moreover, since the components of these adjuvants they lack reactive potential, their suitability for use As adjuvants for vaccines is even greater. It should also be noted that these adjuvants consist of known injectable materials and They appear in commercialized injectables.

The vaccines used in the composition of the invention can be administered in any prescribed manner for the particular vaccine used, preferably by intramuscular or subcutaneous injections. They can be used in veterinary or human vaccines and include inactivated vaccines, both whole and subunits, as well as toxoids. Moreover, the vaccines used are those used to immunize against bacterial, rickettisco and viral pathogens. Suitable human vaccines could include, for example, vaccines, whole and in subunits, against influenza, polio, arbovirus infections, typhoid and paratyphoid fever, ekolcra, plague, pertussis, typhoid, Rocky Mountain fever, Hemophilus influenza type B, pneumococcal multivalent polysaccharide, meningococcal group C, and newly developed vaccines of human diploid cells against rabies and hepatitis vaccine.

Suitable veterinary vaccines could include, for example, vaccines, whole and in subunits, against equine influenza viruses, equine herpes virus, equine encephalomyelitis virus, wart viruses, glossophobia virus, rabies, panleukopenia feline, feline rhinotracheititis, feline calicivirus, infectious bovine rhinotracheititis, paragripe-3, bovine diarrhea virus, bovine adenovirus, pseudorrabia, infectious gastroenteritis virus, porcine parvovirus, canine adenovirus, canine distemper virus. Vaccines whole and in subunits, bacterins and toxoids against equine, brucellosis, vibriosis, leptospirosis, clostridia infections, salmonellosis, colibacillosis, anaplasmosis, Pasteurella infections, Haemophilus infections, erysipelas, and the like. Moreover, it is noted that since it is possible that future vaccines, especially antivirals and antibacterials of the subunit type, are weak immunogens, it will be necessary to potentiate them by means of a suitable and acceptable adjuvant, and it is considered that the system of the present invention It will be very suitable.

The advantages of using the powerful adjuvants described with vaccines are significant. By means of the modulation of certain compartments of the immune system, the adjuvants may cause an increase in immune reactivity of humoral immunity, resulting in an intensification of Antibody production against the antigenic material contained in the preparation of the vaccine with which the adjuvants to the recipient. This intensification will allow to reach, naturally, higher and longer levels of immunity, although the immunization agent is a weak immunogen, such as it can be found in many of the entire inactivated vaccines, and especially those of subunits, and in toxoids. This intensification of the immune response to the antigen has a direct result in this effect, the additional advantage of which is possible to use less antigenic immunization material, thereby decreasing the possibility of serious reactions and host problems against immunization. This is especially important in the vaccination of young people. Moreover, in addition to increase the efficacy and safety of immunizations with a reduced dose of purified antigen, vaccine manufacturing It can be cheaper and more feasible.

The compositions of the invention are prepared dissolving or suspending the adjuvant material in the diluent of antigen and then combining suitable volumes of the solution of adjuvant and antigen solution to antigen dilution appropriate. Antigen diluents are common in the technique, such as phosphate buffered saline, medium Minimum essential, peptone and the like.

The adjuvants of the present invention can mixed together at a concentration higher than the final and subsequently diluted with the vaccine and, optionally, with buffer, before use or storage. It is preferable to use sterile adjuvant components or, in some cases, use the sterile filtration The combination of adjuvant components resulting is then thoroughly homogenized, preferably with a homogenizer at an adequate speed and time, to ensure homogenization of the mixture, for example, at a time from 1 to 10 minutes, before mixing with the chosen vaccine.

More particularly, the adjuvants of the The present invention can be prepared by the following stages:

one)

In First, all components of the adjuvant should be taken to a temperature of about 37 ° C.

2)

 He buffer, if used, the squalene component and the component of Glycerol should be mixed together and let them reach the room temperature. This mixture should be kept at temperature room for about 15 minutes.

3)

 TO the surfactant component is then added to the mixture, and it mixes. This total mixture of adjuvant components can be then sterilized in autoclave and sonicated, to give rise to a homogenous sterile adjuvant mixture. The sonication can take carried out with a probe sonicator of the Heat Systems type set to approximately no 6, in an ice bath. A total sonication time of at least five minutes, dividing the time in sonication pulses of approximately 30 seconds, followed by intervals of 15 seconds in which Keep the adjuvant mixture on ice. As an alternative uniform and preferred to this type of sonication, the mixture of adjuvant can be made microfluidic or homogenized until it passes through a 0.22 µm filter. If desired, the salts of aluminum described herein can then be added to the adjuvant composition, and mix.

4)

 The viral vaccines or vaccine concentrates can then be added to the adjuvant mixture, according to the desired dose.

5)

 The adjuvant / vaccine combination may be mixed slightly to continuation. An additional amount of buffer can be added during mixing, to achieve the volume and concentration of Desired components of the final mixture.

Example 1

Adjuvants of the present invention were prepared and comparative adjuvants and mixed with the trivalent vaccine Wyeth-Ayerst 1993 against the influenza virus (described on page 2578 of the Physician's Desk Reference, 1993, 47th Edition) to prepare the vaccine composition of mammals of the present invention. Injections of the vaccine composition containing 1.5 µg of the hemagglutinin of each of the three strains present in the vaccine, A / Beijin / 32/92, A / Texas / 36/91 and B / Panama / 45/90, to groups of 6 CD-1 mice, female, 7 weeks old. Every mouse received intramuscular injections of 0.2 ml of the vaccine Wyeth- Yesterday. 28 days after inoculation, the blood of the mice and the serum was analyzed by hemagglutination inhibition procedure, looking for antibodies against component A / Beijin. Table I, more exposed Go ahead, present the list of results for each one, expressed as the geometric mean of the value in the 6 mice of each group. It should be noted that the PBS buffer was used in quantity enough to reach the desired volume with the adjuvants and comparative adjuvants described herein.

The vaccine compositions tested contained The following adjuvants. The quantities of the components of the adjuvant are given in% by volume of the vaccine composition final:

Vaccine no 1) 5% squalene, 5% glycerol and 0.2% Tween 80.

Comparative vaccine No. 1) The vehicle for Formulation of Syntex adjuvant, microfluidified, as used at the Syntex Laboratories. This vaccine comprises 5% squalene, 1.25% Pluronic L-121 (marketed by BASF Wyandotte Corp.) and 0.2% of Tween 80.

Comparative vaccine No. 2) 5% squalene, 0.2% Tween 80

Comparative vaccine No. 3) 5% squalene, 1.0% Tween 80

Comparative vaccine No. 4) 5% squalene, 2.0% of Tween 80

Comparative vaccine No. 5) 5% squalene, 4.0% of Tween 80

Comparative vaccine No. 6) 5% squalene, 0.2% of Lecithin, 0.2% Tween 80.

Comparative vaccine No. 7) 5% squalene, 5% glycerol, 0.5% lecithin.

Comparative vaccine No. 8) 5% squalene, 0.2% of Povidone, 0.2% Tween 80.

Comparative vaccine No. 9) the vaccine in PBS buffer, alone (without adjuvant).

TABLE I Comparison of adjuvants against flu

Adjuvants tested IH value (thousands) No. 1 vaccine 2,048 Empty comp. nº one 5,793 Empty comp. nº 2 1,825 Empty comp. nº 3 1,625 Empty comp. nº 4 2,048 Empty comp. nº 5 2,048 Empty comp. nº 6 1.29 Empty comp. nº 7 1,149 Empty comp. nº 8 0.406 Empty comp. nº 9 0.406

Example 2

Additional tests of adjuvants with groups of Cynomolgus monkeys were completed. Each group of 3 monkeys was inoculated intramuscularly with 0.5 ml of the Wyeth-Ayerst 1993 trivalent vaccine against influenza virus (as described on page 2578 of the Physician's Desk Reference, 1993, 47 a) Edition) mixed in 50:50 ratio with the adjuvants described below. On day 28 after the initial vaccination, the monkeys were revaccinated with the same inoculum. 5 ml of blood were taken from each monkey on day 28 and on day 42, for analysis of creatinine phosphokinase (cpk), the results of which are shown below for each group of three monkeys in Table II. As mentioned above, the PBS buffer was used in sufficient quantity to reach the desired volume with the adjuvant / vaccine combinations described below.

Proven Vaccine Compositions

Vaccine A : A vaccine composition of the present invention comprising 5% squalene, 20% glycerol and 0.2% Tween 80, each by volume, of the final composition.

Comparative vaccine A : Vaccine composition containing only vaccine in PBS buffer.

Comparative vaccine B : Vaccine composition comprising 2 mg of cholesterol hemisuccinate liposomes (CHS) or cholesteryl hydrogen succinate, sold by Sigma Chemical Company, St. Louis, Missouri, catalog number C 6013.

Comparative vaccine C : Vaccine composition containing 5% squalene, 30% propylene glycol and 0.2% Tween 80, each by volume, of the final composition.

TABLE II Comparison of adjuvants in Cynomolgus monkeys

Value of IH vs. A / Beijin (GMT)

Vaccine Day 28 Day 42 Vaccine A 203 645 Empty comp. TO 8 8 Empty comp. B 51 102 Empty comp. C 102 256

Claims (18)

1. Mammalian vaccine composition comprising an entire or subunit inactivated vaccine and an effective amount of an adjuvant, characterized in that the adjuvant comprises squalene or squalane, or a mixture thereof, glycerol and a surfactant. 2. Composition of mammalian vaccine according to claim 1, wherein the adjuvant comprises squalene or Squalane or a mixture thereof, in the amount included between 1% and 40% by volume of the total composition, glycerol in the amount between 5% and 40% by volume of the total composition, and a surfactant in the amount comprised between 0.1% and 4.0% of the total composition. 3. Composition of mammalian vaccine according to claim 1, wherein the adjuvant comprises squalene or Squalane or a mixture thereof, in the amount included between 2% and 20% by volume of the total composition, glycerol in the amount between 10% and 30% by volume of the total composition, and a surfactant in the amount comprised between 0.15% and 1.6% of the total composition. 4. Composition of mammalian vaccine according to claim 1, wherein the adjuvant comprises squalene or Squalane or a mixture thereof, in the amount included between 3% and 7% by volume of the total composition, glycerol in the amount between 15% and 25% by volume of the total composition, and a surfactant in the amount comprised between 0.18% and 0.22% of the total composition. 5. Mammalian vaccine composition according any of the preceding claims, wherein the adjuvant further comprises a buffer of acceptable pH from the Pharmaceutical point of view. 6. Mammalian vaccine composition according to claim 5, wherein the pH buffer acceptable from the point Pharmaceutical view is PBS. 7. Mammalian vaccine composition according to any of the preceding claims, wherein the adjuvant further comprises an aluminum salt. 8. Mammalian vaccine composition according to claim 7, wherein the aluminum salt is present in the composition at a concentration in weight / volume between the 0.1% and 2.0%. 9. Mammalian vaccine composition according to claim 7 or 8, wherein the aluminum salt is hydroxide of aluminum. 10. Mammalian vaccine composition according to any of the preceding claims, wherein the vaccine It is a vaccine in subunits against influenza A. 11. Mammalian vaccine composition according to any of the preceding claims, wherein the adjuvant comprises squalene, glycerol and a surfactant. 12. Mammalian vaccine composition comprising an entire or subunit inactivated vaccine and an effective amount of an adjuvant in a pharmaceutically acceptable buffer, characterized in that the adjuvant comprises squalene in the amount between 3% and 7% by volume of the total composition, glycerol in the amount between 15% and 25% by volume of the total composition, a surfactant in the amount between 0.18% and 0.22% of the composition total, and from 0.1% and 2.0% of an aluminum salt. 13. Mammalian vaccine composition according to claim 12, wherein the aluminum salt is hydroxide of aluminum. 14. Adjuvant comprising squalene or Squalane, or a mixture thereof, glycerol and a surfactant 15. Adjuvant according to claim 14, which it also comprises a pH buffer that is acceptable from the point of view pharmacist. 16. Adjuvant according to claim 15, in the that the pharmaceutically acceptable pH buffer It's PBS 17. Adjuvant according to any of the claims 14 to 16, wherein the adjuvant further comprises an aluminum salt 18. Adjuvant according to claim 17, in the that the aluminum salt is aluminum hydroxide.